PROJECT SUMMARY Transgenic animal models are vital for research in biomedical science and have allowed us to elucidate and treat the root causes of numerous human diseases. The overall goal of this project is to develop robust and easy to implement methods to generate genetically edited mouse lines with uniform, non-mosaic genotypes in a single generation. With the advent of genome editing technologies we can now genetically manipulate mouse embryos to make precisely modified animal lines for biomedical research. While genome editing technologies have greatly simplified the process of site-specific genomic manipulation, when applied to embryos, current methods generally fail to uniformly edit all the cells of a developing embryo resulting in a mouse that is genetically mosaic. Furthermore, even when these technologies successfully edit all the cells of an embryo there is no way to detect which embryos are non-mosaically edited. As a result, mice that develop from the gene-edited embryos must be crossed to generate the desired non-mosaic edited mouse line in the next generation. It can take six months or longer to generate a desired mouse line making it time consuming, costly, and labor intensive. Using a combination of the CRISPR-Cas9 system, live imaging, and Adeno Associated Virus Serotype 6 (AAV6), we have developed a method to make site-specific modifications to the mouse genome non-mosaically and to detect non-mosaic, mono-allelic targeted embryos in real time. In this proposal, we will further develop this platform for single-generation transgenic mouse production by (1) optimizing it to achieve the highest rates of non-mosaic targeting possible, (2) developing it to allow for the selection of embryos that have non-mosaic bi-allelic knock- ins, (3) applying it to make specific nucleotide changes in mice, and (4) adapting it to generate a genetic reporter mouse. Due to our ability to track cells of an embryo that have undergone homology-directed repair (HDR) in real time, in the process of completing these aims we will also gain significant insight into DNA double strand break repair dynamics in early mammalian embryos. Successful completion of this research will yield a suite of tools to produce various types of genetic modifications in mouse embryos at high efficiency. By reducing the time, cost, and numbers of mice required to produce new genetically modified mouse lines, this new technology will improve research access to new transgenic research animals and contribute to efforts to understand human diseases and to develop new therapies.